The testing of a large number of molecules in an appropriate biochemical or cellular assay is usually one of the first steps in a drug discovery project [Smith A. Nature 418, 453–459 (2002)]. Screening based on Fluorescence Polarization (FP) [Checovich, W. J. et al. Nature 375, 254–256. (1995); Pope, A. J. et al. Drug Discov. Today 4, 350–362 (1999)], Fluorescence Resonance Energy Transfer (FRET), Homogeneous Time-Resolved Fluorescence (HTRF) and the traditional Scintillation Proximity Assay (SPA) are nowadays the technologies of choice for a biochemical assay [Parker, G. J. et al. J. Biomol. Screening 5, 77–88 (2000)]. The sensitivity and miniaturized format of these methodologies allow for the high throughput screening (HTS) of large proprietary compound collections. However, the complexity of the assays utilized in HTS often results in a large number of hits that are then not confirmed in a subsequent secondary assay. It is now well accepted that for screening, quality is more important than quantity [Smith A. Nature 418, 453–459 (2002)]. Therefore, ways of making the biochemical assays for identification of new lead molecules more reliable are in continual development.
Over the last few years Nuclear Magnetic Resonance (NMR)-based screening has emerged as a powerful tool for lead molecule identification [Hajduk, P. J. et al. Quarterly Reviews of Biophysics 32, 211–240 (1999); Stockman, B. J. et al. Prog. NMR Spectr. 41, 187–231 (2002); Meyer, B. et al. Chem. Int. Ed. 42, 864 (2003)]. Although NMR has been applied extensively for characterizing the products of an enzymatic reaction or to gain insight into the kinetics of the reactions [Percival, M. D. & Withers, S. G. Biochemistry 31, 505–512 (1992)], NMR-based biochemical screening that measures the inhibition or the activation of an enzyme for the identification of new lead molecules has found limited applications [Chiyoda T. et al. Chem. Pharm. Bull. 46, 718–720 (1998)]. The low sensitivity limits NMR to enzymatic reactions with very high substrate concentration. Nonetheless, when NMR is compared to the other techniques utilized in HTS, it provides a more reliable array of data. NMR techniques monitor ligand binding to the receptor via the small molecule signals or the protein signals.
Recent developments using 1H and in particular 19F NMR competition binding experiments have allowed for a reduction of substrate and protein consumption for the screening. In addition, these experiments provide with a single point measurement the value of the binding constant of the identified ligand. Percival and Withers used a NMR method to study binding of an enzyme to its fluorinated substrate analogues [Percival, M. D. & Withers, S. G. Biochemistry 31, 505–512 (1992)]. Such method utilizes 19F NMR detection and requires the presence of a fluorine atom on non-peptide substrates. The 19F signals of the starting and enzymatically transformed substrates are then monitored. Experiments can be performed in real time allowing the observed enzymatic activity to be monitored kinetically or in an endpoint assay format.